Branched-chain sugars and nitrogen-containing sugars are two important classes of naturally occurring carbohydrates. The establishment of these sugars as vital components for the efficacy and specificity of many biologically active natural products has purported the idea that altering and/or exchanging these crucial sugar structures by exploiting their biosynthetic machineries may enhance or vary the physiological characteristics of their parent molecules. Fully realizing the potential of such an approach requires a thorough understanding of the biosynthetic pathway of each target sugar, including genetic, enzymatic, and mechanistic information. Efforts directed toward these goals have achieved some notable results through our work funded by this grant. In the last grant period, we studied the formation of representative branched-chain sugars -- mycarose, yersiniose and galactofuranose, and two amino sugars -- mycaminose, and desosamine. We also extended the scope of this project by exploring the feasibility of generating new glycoconjugates by manipulating the sugar biosynthetic machineries to alter the appended sugars in macrolide antibiotics. As a result of these studies, we have now identified three areas worthy of further in-depth investigation in the next grant period. Outlined in this proposal are our plans (A) to study the mechanism of the formation of dihydrosteptose catalyzed by TDP-dihydrostreptose synthase, (B) to investigate the biosynthesis of an unusual nitrosugar, kijanose, and (C) to exploit the catalytic capabilities of glycosyltransferases in biosynthetic applications. The intended goals of the first two projects are to establish the entire pathway for the biosynthesis of kijanose and to characterize the mechanisms of the C-C and C-N bond formation steps involved the formation of dihydrostreptose and kijanose. These studies will not only aid in delineating how chemical transformations are affected by the responsible enzymes, but will also facilitate future therapeutic design efforts to control and/or mimic their catalytic roles. The insight gained from the third project will allow rational design of hybrid glycoconjugates through the coupling of desired sugars with various aglycons catalyzed by the appropriate glycosyltransferases, harnessing the structural diversity provided by both components. Such a combinatorial approach holds promise for the exploration of new chemical entities that will ultimately be used to battle innumerable microbial threats to human health. Overall, our results are expected to make a significant contribution to the fundamentals of enzyme chemistry and possibly impact pharmaceutical biotechnology.